This year we continued to study the activities of the RAG1-RAG2 protein complex, specifically focusing on the effects of modifications of these proteins. Some years ago, our group showed that the N-terminal region of RAG1 contains a RING finger domain with ubiquitin ligase (E3) activity; this fragment was able to modify a nearby lysine residue (K233) within RAG1. At that time we could not determine the effects of this alteration on the DNA cleavage activity of RAG1/2 , because it was not possible to purify full-length RAG1 (we were using an active form that was missing the N-terminal region). With our present ability to obtain active full-length RAG1, we have returned to studying the effect of ubiquitylation on the DNA cleavage activity of RAG1/2. Extending previous results, now with the ubiquitylated form of the enzyme fully purified, we find that cleavage activity is stimulated by several-fold. Efforts to assess the effect of this modification inside cells are under way. In a separate investigation (in collaboration with Dr. Jay Chung, NHLBI), we found that RAG1 is phosphorylated at a specific site by AMP-activated protein kinase, and that this modification also increases the activity of RAG1/2, as well as increasing V(D)J recombination in cells. It is becoming evident that V(D)J recombination is modulated by several metabolically significant pathways.